Listeria monocytogenes, a foodborne pathogen that can cause serious human disease, is estimated to cause 2,500 cases and 500 deaths annually in the US. Our understanding of the complex mechanisms required for bacterial survival and multiplication throughout foodborne transmission, including in stressful environments prior to infection (e.g., in foods), as well as those encountered in the host (e.g., in the gastrointestinal tract), is limited. Therefore, the long-term objective of this project is to use L. monocytogenes as a model system for exploring mechanisms that contribute to foodborne pathogen transmission and infection, with the goal of reducing the overall public health burden of foodborne diseases. Preliminary data suggest that multiple regulatory elements function in concert to control gene transcription under rapidly changing conditions such as those encountered during foodborne transmission. Thus, the work proposed in this application will test the following hypotheses: (i) networks among key L. monocytogenes regulatory proteins (e.g., PrfA and different alternative C factors, including CB) are necessary for appropriate expression of genes critical for pathogen transmission and virulence in extra- and intra-host environments;and (ii) multiple regulatory networks and stress response systems contribute to gastrointestinal survival and pathogenesis. These hypotheses will be tested through the following five specific aims: (1) Define regulons controlled by the L. monocytogenes alternative sigma factors using full genome microarrays. (2) Identify L. monocytogenes proteins that co-regulate genes contributing to transmission and virulence using selected mutant bacterial strains, microarrays, qRT-PCR and bioinformatics strategies. (3) Characterize global L. monocytogenes gene expression patterns under different environmental stress conditions and in selected intracellular and intra-host environments using microarrays and qRT-PCR. (4) Develop a WWW-based database of L. monocytogenes microarray data and transcriptional profiles. (5) Characterize stress response and virulence phenotypes of L. monocytogenes with mutations in selected genes encoding regulatory proteins and in selected genes regulated or co-regulated by alternative C factors and other transcriptional regulators. Overall, the proposed studies will provide an improved understanding of the mechanisms used by foodborne pathogens to regulate gene expression under rapidly changing environmental stress conditions encountered during transmission and infection. We anticipate that this knowledge will identify mechanisms that can be targeted for development of novel and innovative bacterial control strategies including new antibacterial therapeutics. PUBLIC HEALTH RELEVANCE: The bacterium Listeria monocytogenes causes about 2500 human listeriosis cases and 500 deaths annually in the US, representing about 10% of all US deaths from foodborne illnesses. This project will provide an understanding of the mechanisms used by Listeria monocytogenes (as well as by other bacteria that cause foodborne illnesses) to regulate gene expression under rapidly changing environmental stress conditions encountered during foodborne disease transmission and infection. This knowledge will contribute to identification of specific bacterial mechanisms that can be targeted for development of novel and innovative bacterial control strategies, including new antibacterial therapeutics.